SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

EFFECTS OF PROTEOLYTIC ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The quality of rice wine made from uncooked, unpolished aromatic red rice grain was improved by use of a commercial preparation of acid protease from Aspergillus niger during ethanol fermentation. The fermentation rate of the mash which contained the acid protease was much higher than that of mash that did not contain the preparation of acid protease. The rice wine made from uncooked, polished aromatic red rice, which usually had a less acceptable aroma, was improved by use of the preparation of acid protease, and large amounts of isobutyl alcohol, n-propyl alcohol and ethyl acetate were detected in the resultant rice wine. By contrast, the quality of rice wine made from the bran fraction of aromatic red rice was not improved by the preparation of acid protease. The polished rice fraction of aromatic red rice was affected by the acid protease and the aromatic quality of the rice wine was improved. The aromatic characteristics of red rice wine made from cooked, unpolished aromatic red rice grains, which was rather inferior in terms of both aroma and color, were also improved by the addition of the preparation of acid protease during ethanol fermentation. Thus acid protease has beneficial effects on the production of aromatic red rice wine.     

PRODUCTION OF SHOCHU ON A COMMERCIAL SCALE FROM POST-DISTILLATION SLURRY BY A NEWLY DEVELOPED RECYCLING PROCESS

During commercial scale fermentation for the production of rice shochu using post-distillation slurry in place of the first-stage mash, it was difficult to stir the mash from the first to the third day because of the high viscosity of the mash. Shochu was therefore fermented with a two-step addition of cooked rice, adding rice on the first and third days, to reduce the viscosity of mash during the first few days. Even though the ethanol concentration of the mash after 9 d was 17.2 v/v% and was a little lower than that obtained in laboratory scale tests, the yield of pure ethanol was 473 ml kg^?1 of rice and this value was higher than the average value (450 ml kg^?1) when shochu is made in the standard manner on a commercial scale. The shochu produced on a commercial scale by the newly developed recycling process was very mild and smooth, and received high scores in sensory tests.     

EFFECTS OF THE COOKING PROCESS ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

Fermentation tests were performed with cooked and/or uncooked, polished aromatic red rice (Oryza sativa var. Indica, Tapol) and aromatic red rice bran using compressed baker's yeast and a preparation of glucoamylase produced by Rhizopus, sp. as saccharifying agent. The quality in terms of both aroma and taste of the red rice wine made with uncooked, unpolished aromatic red rice was much higher than that of rice wine made with cooked, unpolished aromatic red rice. However, the bran fraction of aromatic red rice was fairly resistant to the cooking process and this fraction was hardly affected or degraded by the cooking process. Furthermore, the bran fraction improved the quality of rice wine made from mash that contained cooked, polished rice which had a rather lower-quality aroma. In red rice wine brewing, the polished rice fraction of aromatic red rice may suffer from undesirable effects during the cooking process.     

EFFECTS OF β-GLUCOSIDASE ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The pigment in the bran layer of aromatic red rice (Oryza sativa var. Indica, Tapol) is rather stable, has a characteristic red color just like that of grape wine and has a peak of absorbance at 530 nm at an acidic pH. A commercial saccharifying agent, glucoamylase AN-2, produced by Aspergillus niger was fractionated to separate glucoamylase and β-glucosidase activities by column chromatography on CM Sephadex C-50. The red rice wine made from uncooked, unpolished aromatic red rice using the fractionated, β-glucosidase-free preparation of glucoamylase had a characteristic red color. By contrast, red rice wine made with glucoamylase AN-2, which contained β-glucosidase activity, was inferior in color. The red pigment of aromatic red rice wine was decolorized and glucose originating from the red pigment was released by enzymatic digestion with the fractionated preparation of β-glucosidase. The partial decolorization of aromatic red rice wine was ascribed to the enzymatic action of β-glucosidase that was present in glucoamylase AN-2. Thus β-glucosidase activity has an undesirable effect on the brewing of aromatic red rice wine.     

Behaviour of ethyl caproate during the production and distillation of ethyl caproate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐rich rice shochu by adding a cultured broth of a caproic acid‐producing bacterial (CAPB) consortium to the fermentation. When the CAPB consortium containing fermented mash was subjected to vacuum distillation, the distillation efficiency of ethyl caproate was up to ~300%, while the distillation efficiencies of the other flavour compounds and ethanol were <100%. The behaviour of ethyl caproate during the production and distillation of an ethyl caproate‐rich rice shochu was investigated and the results showed that ethyl caproate was synthesized by Saccharomyces cerevisiae during the shochu production process, and that some of the ethyl caproate synthesized was secreted into the medium. Ethyl caproate in the medium evaporated easily and was transferred to the distillate when distilled. The increase in ethyl caproate after distillation was mainly derived from the release of the intracellular ethyl caproate, with negligible input from the chemical esterification of caproic acid and ethanol. During vacuum distillation, although few yeast cells were disrupted, secretion of the intracellular ethyl caproate was more efficient owing to the increase in temperature, allowing the distillation efficiency of ethyl caproate to be >100%. Copyright © 2016 The Institute of Brewing & Distilling     

PRODUCTION AND CHARACTERISTICS OF RED RICE SAKE

Red rice sake was efficiently produced from unpolished red rice without cooking by use of rice koji prepared with shochu koji mold, Aspergillus kawachii, as saccharifying agent. A comparative study was made of red rice sake made with rice koji and red rice wine made with a preparation of glucoamylase known as Sumizyme. Red rice sake contained about 13% ethanol and its acidity was 8.6. Large amounts of volatile compounds, such as isobutyl alcohol and ethyl acetate, were found in the red rice sake. The red color of the red rice sake was more intense than that of the red rice wine. The quality of red rice sake was better than that of red rice wine, as assessed by gas chromatography, spectrophotometric analysis and organoleptic testing. In accordance with the traditional method for sake brewing, red rice, rice koji and tap water were added to the initial mash. The red color of the red rice sake made with these additions was more intense than that of red rice sake made without these additions. The rice wine was somewhat improved by these additions, as assessed by organoleptic testing. The body of the resultant rice wine was fortified with dextrin and its mild characteristics were derived from sugar and dextrin.     

Development of Efficient Shochu Production Technology with Long‐term Repetition of Sashimoto and Reuse of Stillage for Fermentation

In traditional shochu production with a long‐term repetition of sashimoto, the activity of yeast cells decreases, generally resulting in bacterial contamination problems. In this study, a pilot scale study was carried out to demonstrate a technique developed with a laboratory scale test, which improved the activity of the yeast in the first‐stage fermentation and the possibility of reuse of stillage for the fermentation to reduce the quantity of stillage from the distillation by 50%. The yeast cells were activated by aeration and stirring for several hours after sashimoto. The yeast cells maintained a high activity level during the entire test period. The ethanol concentration of the second‐stage fermented mash was improved and an ethanol concentration of more than 17.5% (v/v) was achieved when stillage was reused in place of water for the fermentation. Comparing the flavour compounds of shochu produced by the traditional process with the long‐term repetition of sashimoto, 0.2 ppm of furfural was detected in the shochu produced from the stillage reuse process. No other significant differences were found in the concentrations of the low‐, middle‐ and high‐boiling flavour compounds examined, nor in the concentrations of isoamyl acetate and β‐phenethyl acetate, which are desired flavour compounds in shochu.     

全水稻发酵生产食用酒精工艺研究

该实验通过全水稻组分分析,成功利用全水稻粉配粉浆进行酒母培养,并经液化、同步糖化发酵、精馏后生产食用酒精。结果表明,全水稻发酵成熟醪平均蒸馏酒精度12.35%vol、平均还原糖含量0.18%、残淀粉含量0.59%、残糊精含量0.49%、酸度6.28 °T、挥发酸含量0.17 g/L,满足酒精发酵生产要求。水稻发酵成熟醪进行酒精分离提纯后,废醪液可生产水稻干全酒精糟（DDGS）产品,降低生产成本,增加盈利。     

ETHANOL FERMENTATION OF VARIOUS RED RICES WITHOUT COOKING

With various red rices as raw material, fermentation without cooking was allowed to proceed at pH 4.0 and 30°C for 4 d for production of novel alcoholic beverages. Ethanol fermentation was achieved with various rice grains. In particular, uncooked wild-rice (Zizania aquatica) is reported now for the first time as being used for ethanol fermentation. The final concentration of ethanol achieved during fermentation was 8.8–9.3% (v/v). Rice wine made from aromatic red rice (Oryza sativa var. Indica, Tapol) had a wine-like red color. It was rich in a fruity aroma and had a characteristic sour taste, as revealed by organoleptic testing. Volatile substances formed have been analysed by gas chromatography. Rice wine made from black rice (Oryza sativa var. Indica) also had a wine-like red color and a sour taste. By contrast, rice wines made from red rice (Oryza sativa var. Japonica) and wild-rice were faintly yellow in color and had a sake-like and a somewhat green tea-like flavor, respectively. Although the red pigments of the aromatic red rice and black rice were solubilized during uncooked ethanol fermentation, the pigments of red rice and wild-rice were insoluble and not available as material for brewing of red-colored alcoholic beverages. Aromatic red rice appears to be a suitable material for the production of a wine-like alcoholic beverage.     

Isolation and Characterization of Shochu Yeasts with Superior Brewing Ability from Shochu Mashes

Seven yeasts with superior ability in terms of alcohol fermentation and aromatic ingredient generation were isolated from 272 wild yeasts obtained from shochu mashes of shochu breweries. These seven yeasts were examined for their fermentation ability with rice and sweet potato using small scale of fermentation tests. Moreover, their thermotolerance was evaluated by growth tests and fermentation tests with barley koji. Among the isolated seven yeasts, the shochu yeast “MF062” was superior in the characteristics of fast fermentation, high alcohol production, aromatic ingredient generation and thermotolerance. It was named the “Heisei Miyazaki Yeast”. From the results of analyses of physiological and genetic characteristics of MF062, it was shown to be a Saccharomyces cerevisiae, but it showed different characteristics from the industrial yeasts used in shochu production. Using a sweet potato shochu brewing test, with 60 kg of raw materials and yeast MF062, it was confirmed that a high alcohol production yield and a high quality shochu could be obtained.     

The Formation of β‐Damascenone in Sweet Potato Shochu

The formation of β‐damascenone during shochu manufacture was investigated by quantifying β‐damascenone at each stage of manufacturing. Steamed sweet potato has a low level of free β‐damascenone (0.02–0.1 μg/g). During fermentation, β‐damascenone was produced in small quantities that were degraded by yeast. Thus, the second mash accumulates little free β‐damascenone (approximately 17 μg/L). The concentration profile in the fractionated distillate showed that β‐damascenone was produced during heating. Most β‐damascenone in shochu was formed during distillation, not during steam heating and fermentation. It is suggested that the level of β‐damascenone in shochu could be increased by reducing the pH of the second mash and prolonging the distillation period. Sweet potato cultivars differed in total free and hydrolyzed β‐damascenone content and there was a strong association between each cultivar and its shochu β‐damascenone content. The selection of the sweet potato cultivar is important for determining the quantity of β‐damascenone in a shochu brew.     

超贮稻谷半连续浓醪发酵生产燃料乙醇工艺研究

该文以脱壳超储稻谷（全糙米）为原料,开展了半连续浓醪发酵生产燃料乙醇工艺研究。在中粮安徽30万t/年半连续燃料乙醇装置上进行了高浓拌料、喷射液化、同步糖化半连续发酵、精馏后制备乙醇等一系列试验。结果表明,全糙米发酵成熟醪平均酒份达到15.36%vol、平均还原糖含量0.21%、残淀粉含量0.40%、残糊精含量0.55%、酸度8.43 °T、挥发酸含量0.35 g/L。该技术在国内处于领先水平,可为企业带来经济效益,值得进一步推广应用。     

Characteristic odour compounds in shochu derived from rice koji

Shochu, a traditional Japanese distilled liquor, makes use of rice koji, which is koji mould grown on rice grain. Rice koji is an essential ingredient of Japanese liquors such as shochu, and it plays a role as a source of the enzyme to degrade starch. However, there has been no research on the effect of rice koji on the flavour of shochu. Therefore, in this study, the volatile compounds in shochu derived from rice koji were investigated. Two shochu samples were prepared to assess the contribution of rice koji to the flavour. One shochu sample was prepared from rice koji, yeast and water (rice koji‐shochu). The other shochu sample was obtained from steamed rice and various enzymes instead of rice koji (enzyme‐shochu), along with yeast and water. The volatile compounds were analysed by gas chromatography–mass spectrometry (GC‐MS) and GC‐MS/olfactometry with aroma extract dilution analysis. The results showed that enzyme‐shochu had a higher flavour dilution value of dimethyl trisulphide and hexanal, whereas rice koji‐shochu had a higher flavour dilution value of some ester compounds that imparted aromatic odours such as fruity. Some unknown peaks representing compounds that impart characteristic odours such as soda, potato, lavender, and tea‐like were specifically detected in rice koji‐shochu. The concentrations and calculated odour active values of 14 compounds were measured. These results showed that isovaleraldehyde, ethyl caprylate, ethyl caproate and ethyl 2‐methylbutyrate played an important role in imparting the specific odour of rice koji‐shochu. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of fermentation by Bacillus subtilis on antioxidant and cytotoxic activities of black rice bran

Black rice bran was fermented with Bacillus subtilis KU3 isolated from Korean traditional food, Kimchi. Antioxidant and cytotoxic activities of the fermented black rice bran were investigated. Total phenolic and anthocyanin contents decreased from 171.54 mg GAE g^?1 and 2.31 mg g^?1 to 139.13 mg GAE g^?1 and 2.12 mg g^?1, respectively, after fermentation. Antioxidant activities determined by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging, β‐carotene bleaching and ferric thiocyanate assay were correlated with total phenolic and anthocyanin contents. Non‐fermented black rice bran extract (NFBE) showed greater antioxidant activities than fermented black rice bran extract (FBE). Cytotoxic activities measured by MTT assay showed that both NFBE and FBE had over 50% activities. The cytotoxic activities of FBE against MCF‐7 and HeLa cells were 71.65% and 68.07%, respectively, at 8.0 mg mL^?1, but those of NFBE were lower than 50%. These results suggested that the cytotoxic activity of black rice bran improved through fermentation, while antioxidant activity reduced.     

Phenolic Profile and Antioxidant Activity of Extracts Prepared from Fermented Heat‐Stabilized Defatted Rice Bran

Heat‐stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value‐added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (‐)‐epicatechin, 4.08 mg p‐courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p‐courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.     

Effects of rice bran fermented with Lactobacillus plantarum on palatability,volatile profiles,and antioxidant activity of brown rice noodles

Brown rice noodles (RN) are typical whole-grain products with health benefits. This study investigated the effects of rice bran fermented with Lactobacillus plantarum on palatability, volatile profiles, and antioxidant activity of brown RN. Three kinds of RN, including RN, brown RN (BRN), and bran fermented RN (BFRN), were involved in this study. The results indicated that bran fermentation reduced the cooking loss of BFRN and improved the elongation and the sensory attributes of brown RN. After fermentation, aldehydes were significantly decreased while alcohols increased, and more pleasant flavours were shown in BFRN. Fermentation increased the content of free phenolics and decreased the content of bound phenolics. Among the four major phenolics detected in this study, ferulic acid was the most abundant one in all three RN samples. The DPPH radical scavenging activity and the ferric reducing antioxidant power of free phenolics in BFRN increased significantly compared to BRN. This study proved that rice bran fermentation is an effective approach to improve palatability, enrich the flavour as well as enhance the antioxidant activity of brown RN, which may facilitate the development of processing technology for brown RN.     

预糖化米酒发酵工艺研究

针对传统米酒发酵工艺的创新升级以及大罐发酵的需求,对米酒发酵过程大米熟料与生料预糖化发酵等进行了深入探究。结果表明,原料经过蒸煮糖化后发酵,发酵醪还原糖含量升高至2倍左右,蒸馏液酒精度提升了10.62%,酒精度达到接近峰值的时间提前1～2 d,但是总酸、总酯含量都有所降低;熟料制浆糖化后酒精度进一步提高9.31%,总酯含量提高56.71%,但总酸略有降低;生料粉碎糖化酒精度进一步提升8.61%,但总酯含量相对减少21.47%。生料粉碎糖化发酵节省能源,发酵醪分散均匀、流动性好,在大罐发酵方面有着广阔的应用前景。     

稻谷加工工艺改进

为探究酵母发酵对米糠风味以及营养特性的影响。本研究采用顶空固相微萃取-气相色谱-质谱技术鉴定不同发酵时期（0、12、18、24、30 h）米糠的挥发性风味物质,通过正交偏最小二乘分析确定影响米糠发酵前后风味的关键性挥发性物质,最后对发酵米糠进行了综合感官评价并比较了发酵最佳时间对其营养组成变化。结果表明：发酵过程中米糠的挥发性物质含量和种类发生明显变化,未发酵米糠中醛类含量占比35.99%,醇类含量占比14.21%,发酵30 h醛类含量下降到5.52%,醇类含量提高到60.87%。其中关键性挥发性物质共15种。在感官评价方面,关键挥发性物质与米糠香气之间有显著相关性（P<0.05或P<0.01）,发酵后米糠感官评分提高,发酵18 h的米糠感官评分最佳。壬醛、壬酸乙酯对米糠的综合评分影响最为显著（P<0.01）。在营养组成方面,酵母发酵米糠粉中的蛋白质含量提升了38.41%,纤维含量提升了18.21%。说明酵母发酵不仅能有效改善米糠风味,还能够提高米糠粉作为食品原料的营养特性。

     

Characteristics of aromatic compound production using new shochu yeast MF062 isolated from shochu mash

The effect of temperature on the production of aromatic compounds using Heisei Miyazaki yeast MF062 was compared between 10 industrial yeasts. All yeasts tested produced characteristic patterns of alcohols and esters in fermentation tests with rice‐koji at 20, 28 and 38°C. The concentration and composition in mature moromi with rice‐koji at 20, 28 and 38°C were almost the same as those with barley‐koji. Therefore, it was suggested that fermentation temperature is an important factor in the production of aromatic compounds. MF062 produced almost the same concentration of β‐phenethyl alcohol at both 38 and 28°C. The concentration was higher than that generated by the other 10 yeasts. MF062 produced higher concentrations of i‐butyl alcohol than the other yeasts at higher fermentation temperatures. Moreover, compared with the other yeasts, MF062 produced a lower concentration of acetate, which can give an off‐flavour in excess concentrations in shochu. The production of acetoin was divided into two groups – a high producing group and a low producing group – at all temperatures. MF062 belonged to the latter group and showed preferred characteristics in the production of shochu, resulting in a high concentration of preferred aromatic compounds and a low concentration of compounds that impart an off‐flavour. Copyright © 2013 The Institute of Brewing & Distilling